Part:BBa_K1074011

PsdpRI+RBS+SDP

We construct this gene circuit as our kill switch to kill the engineered Bacillus subtilis for the safety purpose.Killing is mediated by the exported toxic protein SpbC. Extracellular SpbC induces the synthesis of an immunity protein, SdpI, which protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor, SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SpbC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein. The kill switch is based on a high-copy vector fused with promoter for operon sdpIR and coding sequence for protein SpbC. When SpbC toxins are sensed, they will be captured by Immunity Protein SdpI at the membrane, enabling SdpI to sequester SdpR. As a result, repression on promoter SdpIR is released and more SpbC will be produced. Trapped in this endless loop, the SpbC producing cells fails to cope with enormous toxin SpbC and doomed after eliminating their siblings. Eventually, the group of engineered Bacillus subtilis is destroyed instead of sporulating.

Usage and Biology

We have built model to verify the validity of this part, and our experiment is still going on.

There are both positive and negative feedback loops in this process. On the one hand, SdpI is unable to sequestrate the autorepressor, SdpR, until it captures the toxin, SpbC. The accumulation of SpbC will thus facilitate SdpI to capture more SdpR and thereby relieve the repression of SdpR, stimulating the expression of itself. This is the positive feedback loop which leads to the increasing accumulation of SpbC and finally the death of the bacteria. On the other hand, the removal of SdpR also enhance the expression of SdpI and accelerate the sequestration of SpbC, which forms a negative feedback loop whose effects contradict the positive feedback loop. However, since the copy number of SpbC is much higher, it is believed that the positive loop is strong enough to outweigh the negative one, which guarantees this mechanism will finally leads to collapse instead of equilibrium.

Results

In engineered bacterial the concentration of SpbC will rise up as the result of positive feedback, killing all engineered bacterial .

In wild bacterial the concentration of SpbC will limited to a certain degree to avoid kill itself .

For more details, refer to our wiki [http://2013.igem.org/Team:USTC_CHINA/Modeling/KillSwitch]

References

Parallel pathways of repression and antirepression governing the transition to stationary phase in Bacillus subtilis AV Banse, A Chastanet, L Rahn-Lee…,PNAS ,2008

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 98
  Illegal SpeI site found at 680
  Illegal PstI site found at 2107
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 98
  Illegal SpeI site found at 680
  Illegal PstI site found at 2107
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 98
  Illegal SpeI site found at 680
  Illegal PstI site found at 2107
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 98
  Illegal SpeI site found at 680
  Illegal PstI site found at 2107
  Illegal AgeI site found at 867
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 854